Mediator profiles in tears during the conjunctival response induced by allergic reaction in the nasal mucosa.

BACKGROUND
The allergic reaction occurring primarily in the nasal mucosa can induce a secondary conjunctival response of an immediate (SICR), late (SLCR), or delayed (SDYCR) type in some patients with allergic conjunctivitis (AC).


OBJECTIVES
To investigate the concentration changes of histamine, tryptase, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), leukotrienes (LTB 4, LTC4, LTE4), myeloperoxidase (MPO), interferon-γ (IFN-γ), and interleukins (IL-2, IL-4, IL-5) in tears during the SICR, SLCR, and SDYCR.


METHODS
In 32 patients with AC, 11 SICR (p<0.01), 13 SLCR (p<0.001), and eight SDYCR (p<0.01) to nasal challenges with allergens (NPTs), the NPTs and 32 control tests with PBS were repeated and supplemented with the determination of these factors in tears.


RESULTS
The SICRs were associated with significant concentration changes in tears (p<0.05) of histamine, tryptase, ECP, LTC4, and IL-4. The SLCRs were accompanied by significant changes in concentrations of histamine, ECP, LTB4, LTC4, MPO, IL-4, and IL-5. The SDYCRs were associated with significant concentration changes in tears (p<0.05) of LTB4, MPO, IFN-γ, and IL-2. No significant changes in these factors were recorded in tears during the 32 PBS controls (p>0.1) or in the ten control patients (p>0.1).


CONCLUSIONS
These results provide evidence for causal involvement of nasal allergy in some patients with AC, inducing secondary conjunctival response of immediate (SICR), late SLCR, or delayed SDYCR type, associated with different mediator, cytokine, and cellular profiles in the tears, suggesting involvement of different hypersensitivity mechanisms. These results also emphasize the diagnostic value of nasal allergen challenge combined with monitoring of the conjunctival response in some patients with AC.

our Department of Allergology & Immunology (Institute of Medical Sciences "De Klokkenberg," Breda, The Netherlands) during 1998-1999 for more extensive analysis of their AC complaints, and developing the secondary conjunctival response (SCR) to nasal provocation tests with allergens (NPTs), volunteered to participate in this study. These patients, 13 men and 19 women, 20-43 years of age (Table 1), had previously been treated with various topical and oral H1-receptor-antagonists, topical cromolyn, glucocorticosteroids, decongestant and vasoconstrictors and incidentally with non-steroidal anti-inflammatory drugs (NSAID), however, with only partial and not fully satisfactory therapeutic effects. None of these patients had other ocular disorders, infection, systemic disease, or immunodeficiency, or had previously been treated with nasal or systemic glucocorticosteroids, nasal cromolyn, or immunotherapy. All of them demonstrated normal intraocular pressure. In 15 of these patients, 19 conjunctival provocation tests (CPTs) with inhalant allergen, performed previously, were negative. The patients underwent a routine diagnostic procedure consisting of a detailed disease history, physical examination, basic laboratory tests, bacteriological screening of tears, nasal secretions, sputum and blood, roentgenogram of chest and paranasal sinuses in Water's projection, nasoscopy, cytologic examination of nasal secretions, skin tests with inhalant and food allergens, determination of serum immunoglobulins, and ophthalmologic examination including ophthalmoscopy, slit-lamp evaluation, vital staining with fluorescein, and cytologic examination of the tears.
The ocular signs and relevant subjective symptoms were evaluated with Pelikan's scoring (grading) system [7][8][9][10]. The patients were investigated in a period without acute ocular and nasal complaints, during hospitalization. The long-acting H1-receptor antagonists, topical cromolyn and glucocorticosteroids, were withdrawn 4 weeks, whereas other treatments were withdrawn 48 h before each NPT. The 32 positive NPTs producing an SCR of any type and 32 PBS control challenges were repeated 2-3 weeks later. The repeated NPTs and PBS controls were supplemented with tear collection for the mediator determination. A 4-day interval was always inserted between the end of the preceding test and the beginning of the following test to prevent carry-over effects and to allow for patient to recover. The study protocol was approved by the local ethical committee (IRB-MCK), and informed consent was obtained from all study participants. The study has been performed according to the WMA Declaration of Helsinki concerning the principles for medical research involving human subjects.
Allergens: Dialyzed and lyophilized allergen extracts (Allergopharma, Reinbek, Germany) were diluted in PBS and used for skin tests in concentrations of 100-500 BU/ml and for NPTs in concentrations of 1000-5000 BU/ml (Table 2), as recommended by the manufacturer.
Skin tests: Skin prick tests (SPTs) with allergenic extracts in concentrations of 500 BU/ml were performed and evaluated after 20 min. The intracutaneous tests in concentrations of 100 BU/ml and 500 BU/ml were then performed in all patients and evaluated 20 min and 6,12,24,36,48,56,72, and 96 h after the intradermal injection. A skin wheal (>7.0 mm in diameter) occurring after 20 min was qualified as a positive immediate skin response, skin infiltration appearing between 6 and 12 h as a late skin response, and skin induration recorded later than 48 h as a delayed skin response [6][7][8][9][10].
Nasal provocation tests: Nasal challenges with allergens were performed using rhinomanometry, described in our previous studies [5][6][7][8][9][10]24]. Nasal obstruction due to the nasal mucosa edema was evaluated by nasopharynx-nostril pressure gradient (NPG) parameters, which are the pressure differences (∆P) between the nasopharyngeal cavity and the outside air, expressed in cm H 2 O. NPTs were performed using the following schedule: (1) baseline values recorded at 0, 5, and 10 min; (2) PBS control values recorded at 0, 5, and 10 min after a 3-min application of PBS to the nasal mucosa of the non-intubated nasal cavity by a saturated wad of cotton wool on a nasal probe inserted under the middle turbinate; (3) the post-challenge values recorded after a 3-min challenge with the allergen, performed in the same manner as the challenge with PBS, at 0, 5,10,20,30,45,60,90, and 120 min, then every hour up to the 12 th h, and every second hour during the time periods between the 24 th -38 th and 48 th -56 th (60 th ) h ( Figure 1) [5][6][7][8][9][10]24]. The allergens used for the NPTs were chosen with respect to the disease history and positive skin tests (Table 1 and Table 2).
Control challenge with phosphate-buffered saline: The control nasal challenge with PBS was performed in each patient studied by the same schedule as that used for the NPTs with allergen.
Collection and processing of tears for mediator measurements: The tear specimens (80-150 μl) were gently collected with a sterilized micropipette from each eye separately from the inferior conjunctival fornix and/or lacus lacrimalis at the following time intervals: (a) at 10, 5, and 0 min before the NPT and (b) at 0, 5, 10, 20, 30, 60, and 120 min and every second hour up to 12 h and between the 24 th -38 th and 48 th -56 th (60 th ) h after the allergen challenge ( Figure 1). The tear specimens collected before the NPT, those collected up to 10 min, and those obtained at 20 and 30 min after the NPT were pooled to obtain sufficient material quantity. Care was taken to avoid touching the eye lid margins, corneal surface, and conjunctiva, as far as possible. Immediately after collection, the tear samples were divided into two equal portions; one portion was processed for cytologic examination, whereas the other portion was centrifuged at 1000 × g for 1 min at 4 ○ C.
The supernatants were removed and stored at −8 °C.
The factors in tears were measured by commercial kits, according to the manufacturer's recommendations. The measurements were performed in tear samples from each eye separately on each occasion, in duplicate and by a double-blind schedule. The results were then calculated as the mean of both eyes. The intra-assay and the inter-assay coefficients of variations for the assay kits were less than 10%. The detection limits measured by the author are abbreviated as DL. The following factors were recorded: (a) Histamine concentrations, so-called blanks, were measured by Siraganian's fluorometric method [28], Collection and processing of tears for the additional cytologic examination: The tear specimens were spread out on the slide surface with a glass probe, air-dried, fixed with polyethylene glycol, and stained with May-Grünwald-Giemsa, which we modified [7,23,29,30]. Specimens were then dehydrated with methyl alcohol, mounted in Canada balsam, and scanned microscopically in a double-blind manner [7,23,29,30]. The particular cell types were distinguished according to the cytologic criteria described in detail in our previous papers [7,23,29,30]. The absolute numbers of particular cell types were counted per microscopic field at magnification 250x and the mean values calculated from 20 fields, per each eye separately. The mean values from both eyes were finally calculated and expressed in numbers of cells per 1 microscopic field (250x magnification). Doubtful cells were reexamined under oil immersion (magnification 1200x).
Control group: Ten randomly selected adults suffering from allergic rhinitis without history of ocular disease and with normal ophthalmologic findings volunteered to participate as control subjects. In these patients, 10 positive NPTs with inhalant allergens (four INR, four LNR, two DYANR) were repeated and supplemented with registration of the conjunctival and subjective symptoms and a single determination of these mediators in tears (Table 1).   Table 2). The 32 PBS control tests were negative (p>0.1).

Nasal responses:
No significant differences were found in the appearance of the particular NR types regarding the individual allergens (p>0.1). The repeated NPTs resulted in the development of similar NR types ( Figure 2C, Figure 3C, Figure 4C). No statistical significant differences were found between the initial and repeated responses (p>0.2).
No significant differences in the conjunctival features recorded during the initial and the repeated SCRs were observed between the right and left eye (p>0.1). No significant corneal signs were recorded in any SCR type. No conjunctival changes were recorded during the 32 PBS control challenges (p>0.1).

Changes in mediators and other factor in the tears during the secondary conjunctival responses:
The SICRs were associated with significant concentration changes (p<0.05) of histamine, tryptase, ECP, LTC 4 , and IL-4 in the tears (Table 3; Figure 2A), whereas the concentrations of EDN, LTB 4 , LTE 4 , MPO, IFN-γ, and IL-2 were either under the detection limits or unchanged. The SLCRs were accompanied by significant changes in the concentrations (p<0.05) of histamine, ECP, LTB 4 , LTC 4 , MPO, IL-4, and IL-5 (Table 4; Figure 3A), but the concentrations of tryptase, EDN, LTE 4 , IFN-γ, and IL-2 were under the detection limits or unchanged. The SDYCRs were associated with significant concentration changes (p<0.05) of LTB 4 , MPO, IFN-γ, and IL-2 in the tears (Table 5; Figure  4A), while the concentrations of histamine, tryptase, ECP, EDN, LTC 4 , LTE 4 , IL-4, and IL-5 were under the detection limits or unchanged, The prechallenge concentrations of most factors were either low or under the detection limit (Table 1), whereas the post-challenge concentration changes appeared during the particular SCR types. No significant concentration changes in these factors were recorded in tears during the 32 PBS controls or 29 negative CRs. No significant differences in the concentration changes of the particular factors in tears were found between the right and left eye, during the SCRs and during the PBS controls (p>0.1 and p>0.2, respectively).
Cytologic changes in tears during the particular secondary conjunctival response types: The prechallenge cell counts in tears were low compared with the primary types of AC [5,7] The SICR was associated with increased counts of eosinophils and mast cells in tears between 30 and 60 min and of the epithelial cells between 60 and 90 min after the NPT   ( Table 6). The SLCR was accompanied by increased counts of eosinophils between 4 and 8 h, neutrophils between 6 and 9 h, basophils between 3 and 4 h, lymphocytes between 10 and 11 h, goblet cells at 10 h, and epithelial cells between 7 and 9 h after the NPT ( Table 7). The SDYCR was associated with increased counts of neutrophils between 24 and 32 h, lymphocytes between 24 and 34 h, monocytes between 12 and 30 h, goblet cells between 34 and 36 h, and epithelial cells between 26 and 34 h after the NPT (Table 8). No significant cellular changes were found in tears of the 32 PBS controls, and only sporadic epithelial cells were recorded in tears during the 29 NNRs. No significant differences were found in results between both the eyes.

Control patients:
The ten control subjects demonstrated neither conjunctival signs nor significant concentration changes in the factors in tears during the ten repeated positive nasal responses (p>0.2).

DISCUSSION
The conjunctiva and the nose have a manifold anatomic and functional relationship [2,3,[7][8][9][10]. The conjunctiva communicates with the nasal cavity through the nasolacrimal duct facilitating tear drainage into the nasal cavity, and through the blood vessel, lymphatic, and neurogenic networks.
Allergic reactions taking place primarily in the nasal mucosa due to intranasal exposure to an inhalant allergen may affect the conjunctiva and other ocular tissues, such as the cornea, in various ways and upon involvement of various mechanisms [7][8][9][10]20,[31][32][33][34]. These mechanisms may include the following: (1) Various cell types participating in the nasal allergic reaction can migrate into the bloodstream and/or lymphatic system and sometimes into lacrimal ways, and thus attain the conjunctiva. (2) Various factors (classical mediators, eicosanoids, cytokines, chemokines, adhesion molecules, and other factors) released during the allergic reaction in the nasal mucosa can reach the conjunctiva either directly by retrograde penetration through the lacrimal ways or indirectly through the related blood or lymphatic vessel system. (3)The nasal allergic reaction can also activate the local neurogenic system (sensory nerves, sympathetic and parasympathetic fibers) releasing the neuropeptides that reach the conjunctiva through related nerves, such as the nervus trigemini, nervus nasociliaris, and ganglion pterygopalatinum. (4) The nasal allergic reaction can also stimulate the local nasal mucosal lymphatic system called the nose-associated lymphatic tissue (NALT), a part of the mucosa-associated lymphatic system (MALT). The MALT system facilitates multiple mutual communication among the particular lymphatic organ-related sub-systems, such as the NALT and the eyeassociated lymphatic tissue (EALT), conjunctiva-associated lymphatic tissue (CALT), tear-associated lymphatic tissue (TALT), and lacrimal drainage-associated lymphatic tissue (LDALT). The manifold communication among the individual lymphatic system parts results in transmission of various cell-cell, cell-receptor, and receptor-receptor signals as well as reciprocal traffic of various circulating cell types, such as plasma cells/B lymphocytes producing immunoglobulins of individual classes and sub-classes, particular sub-sets of T-lymphocytes (Th1-and Th2-cells, cytotoxic, regulatory, and natural killer cells), antigen-presenting cells (APCs), and other cell types [8][9][10]20,[29][30][31][32]. The cell traffic can be realized not only through various attraction mechanisms governed by chemotactic factors, cytokines, chemokines, and adhesion molecules but also through the homing mechanism of B-and T-lymphocytes, controlled by several homing factors [8][9][10]20,[31][32][33][34]. The disturbed homing mechanism leads to migration of particular cell types (e.g., B-or T-lymphocytes) to locations different from the predetermined destinations. The particular lymphocyte subsets having been initially activated in a certain tissue, after migrating into the blood and/or lymphatic stream to finish their maturation process, do not return to this original tissue, but terminate their route in another tissue. This process is called wrong homing [8][9][10]20,[31][32][33][34].
Generally, there is a dearth of structural data demonstrating the course of mediator and cytokine profiles in tears during the particular types of the primary conjunctival response to the CPT. Data illustrating the appearance and concentration changes of mediators and cytokines in tears during the secondary forms of conjunctival response to NPTs are, unfortunately, not available. Moreover, few data are also available on the mediator and cytokine appearance and profiles in the tears of healthy subjects, which may serve as reference values [58,59].
Our results demonstrating increased concentrations of histamine, tryptase, ECP, LTC 4, IL-4, and IL-5 in tears during the SICR would suggest the involvement of mast cells, eosinophils, and probably of Th 2 -lymphocytes in the mechanism  underlying this response type. The concentration changes in histamine, ECP, LTB 4 , LTC 4 , MPO, IL-4, and IL-5 in tears during the SLCR may be suggestive of the role of basophils, eosinophils, neutrophils, and Th 2 -lymphocytes in SLCR, whereas the concentration changes of LTB 4 , MPO, IFN-γ, and IL-2 in tears during the SDYCR may be indicative of the involvement of neutrophils, monocytes, and Th 1 -lymphocytes in this response type. However, this presumption has also some limitations since various mediators and cytokines can be produced by various cell types. Moreover, these mediators have most probably been released by cells participating in the primary allergic reactions in the nasal mucosa. This hypothesis is supported by our previous results, demonstrating only limited numbers of these cell types, in a non-activated condition, in tears during the secondary conjunctival response types [7][8][9][10]23,56].
Another important aspect concerns the participation of the cells in an immunologic process. The appearance of a certain cell type in a medium, in this case in the tears, does not necessarily signify its activation and active participation in that immunologic process but can also be a consequence of another, proceeding, mechanism or its part. Vice versa, the activation of a particular cell type and its active participation in an immunologic process may not necessarily result in the changes of its count (e.g., increase).
The simultaneous recording of the concentration changes of particular mediators and some cytokines in the tears and of the changes in the counts of individual cell types in the Table 6. mean numbers of parTiCular Cell Types in Tears (per miCrosCopiC field aT 250×magnifiCaTion) during The posiTive siCr and pbs Challenge (n=11). However, the results of the current study are limited only to the tears and cannot serve as an indication of the cellular and mediator changes and processes in the conjunctival mucosal membrane. These changes can be demonstrated only by biopsies [11,14,16]. This fact may be regarded as a limitation of the current study. The lack of measurements of the particular mediators and other cytokines in the nasal secretions simultaneously to their recording in the tears, which had not been performed due to technical reasons, is also a limitation of this study. In addition, the variation of the Table 7. mean numbers of parTiCular Cell Types in Tears (per miCrosCopiC field aT 250×magni-fiCaTion) during The posiTive slCr and pbs Challenge (n=13). tear sample volumes may be considered as a certain deficit of this study. The results of the current study may therefore be assessed more as a trend of the individual mediator profiles in tears during the particular types of the secondary conjunctival response. A simultaneous measurement of some tear components, such as total protein, lysozyme or lactoferrin, indicating the tear dilution degree, would improve the quantification of investigated factors in tears in further studies. Similarly to all studies in tears, the exact differentiation of the basal portion of tears resulting from the immunologic mechanism(s) from the tear portion caused by the reflex mechanism during collection of the material is a difficult if not impossible technical problem. Nevertheless, we believe that the tear samples collected by a careful and non-irritating technique used in the current study, have been produced by the genuine immunologic mechanism. Even if some negligible reflex mechanism, in despite of gentle tear collection, has induced a marginal amount of reflex tears, then it was of the same range throughout the whole study, and thus without further significant influence on the study results.

Cells
Finally, the results of the current study may also have an important impact on the therapeutic management of these ocular disorders, regarding not only the choice of the most suitable drugs but also the administration route [5][6][7]10,23,[61][62][63]. However, further investigations, such as biopsy combined with immunohistochemical methods and flow cytometry of the conjunctival and adjacent tissues, will be necessary to clarify fully the mechanism(s) underlying the primary as well as the secondary conjunctival response types.